1. Field of the Invention
The invention relates to the use of natural product compounds isolated from plants, and synthetic chemical analogues thereof, for the prevention and treatment of beta-Amyloid protein-induced disease. More particularly, the invention relates to pharmaceutical compositions that protect neuronal cells from beta-Amyloid insult for use in preventing and treating beta-Amyloid protein-induced disease.
2. Description of Related Technology
Alzheimer's disease (AD) is the most common cause of progressive cognitive dysfunction. AD affects approximately four million Americans and causes more than 100,000 deaths each year, with a total annual cost approaching $70 billion. It is estimated that by the year 2020, 14 million Americans will be affected by the disease. Furthermore, AD has a profound effect on the millions of family members and other loved ones who provide most of the care for people having this disease. Unfortunately, the cure for AD has not been discovered.
The principal pathological characteristics of AD are senile plaques and neurofibrillary tangles (NFT's). Senile plaques are extracellular deposits, principally composed of insoluble aggregates of beta-Amyloid protein (βA protein), that are infiltrated by reactive microglia and astrocytes. Plaques are diffusely distributed throughout the cerebral cortex of AD patients, and are the neuropathologic hallmark of the disease. These plaques or fibril deposits are believed to be responsible for the pathology of a number of neurodegenerative diseases including, but not limited to, Alzheimer's disease. NFT's are intraneuronal accumulations of paired helical filaments, composed mainly of an abnormal form of tau protein, a microtubule associated phosphoprotein which can promote microtubule formation. In the AD brain, the tau protein in NFT is hyperphosphorylated, a condition which has been suggested to contribute to the destabilization of the microtubule network, thereby impairing the axonal network, and eventually causing neuronal death. NFT's occur primarily in medial temporal lobe structures (hippocampus, eniorhinal cortex, and amygdala), and NFT density appears to correlate with dementia severity.
Senile plaques and NFT's appear to be involved in cerebral amyloid angiopathy, consequent neuronal loss, and cerebral atrophy leading to dementia. Although research findings suggest that both plaques and NFT's are involved in disrupting nerve cell functions, the mechanisms that lead to the pathology are not clearly understood.
β-Amyloid peptide has been suggested as one of the major causes of AD. βA peptide (25-35) was shown to exert direct toxic effects on neurons and to inhibit neurite growth in vitro in a dose dependent manner. See Meda et al., Nature 374,647 (1995) and Lamer, Neurosci. Res. Commun. 20, 147 (1997). Thus, therapeutic approaches that can modulate βA peptide toxicity have been hypothesized to represent important methods for controlling the onset of AD. It is envisioned that if neuronal cells can be protected from βA peptide/senile plaque-induced toxicity, the onset of AD may be delayed or prevented. Current pharmacological approaches related to AD preventive and neuroprotective interventions include nicotinic and muscarinic agonists, estrogen, calcium channel blockers, Zinc, sulfonated compounds, triaminopyridine nonopiate analgesic drugs, and non-steroidal anti-inflammatory drugs such as ibuprofen and aspirin. Of particular interest to the present invention is the observation that an anti-βA protein antibody was shown to clear senile plaques and protect mutant PDAPP mice from the onset of AD. See St. George-Hyslop et al., Nature 400, 116 (1999).
The generation of reactive oxygen intermediates through oxidative stress caused by βA peptide has been suggested to be the major pathway of βA peptide-induced cytotoxicity. Senile plaques have been shown to exert a cytotoxic effect on neurons by stimulating microglia to produce reactive oxygen species (ROS). The damaging effect of ROS can be prevented by the free radical scavenging enzyme superoxide dismutase.
Aging of synthetic βA peptide (1-42) for 7 to 14 days at 37° C. in modified Eagle's media was also demonstrated to cause neurotoxic free radical formation. However, aging βA peptide (1-42) in the presence of the media supplement B27, which contains antioxidants as well as other agents that provide protection against oxidative damage, has been shown to inhibit βA peptide-induced neuroloxicity. Nonetheless, while antioxidants such as propyl gallate, Trolox, Probucol, and Promethazine provide significant protection against oxidative insults, they do not protect against βA peptide insults. Furthermnore, it has been shown that βA peptide-induced lipid peroxidation does not contribute directly to cell death.
When the neuroprotective effect of lazaroids (U74389G and U83836E), 21-aminosteroids with antioxidant activity, were tested on cortical cells grown with or without fetal calf serum, U74389G did not protect the cells from βA peptide (25-35) insult in either condition, while low concentrations (15 nM) of U83836E protected the cells exposed to βA peptide in the presence of fetal calf serum. See Lucca et al., Brain Res., 764, 293 (1997). These data suggest that the primary mechanism by which these compounds protect cells from βA peptide-induced toxicity may not involve antioxidative pathways.
In addition to βA peptide-induced ROS mediated neurotoxicity, βA peptide has been shown to cause neuronal cell death by stimulating microglial expression of tumor necrosis factor a (TNFα). The accumulation of βA peptide as neuritic plaques is known to be both trophic and toxic to hippocampal neurons, causing apoptosis or necrosis of the neurons in a dose dependent manner. βA peptide was demonstrated to induce these cellular effects by binding with a receptor for advanced glycation end products (RAGE) that was previously known as a central cellular receptor for advanced glycation endproducts. RAGE was suggested to mediate the interaction of βA peptide with neurons and with microglia, resulting in oxidative stress mediated cytotoxicity. Blocking RAGE with anti-RAGE F(αβ′)2 prevented the appearance of TNFα messenger RNA and diminished TNFα antigen to levels seen in untreated cells. Thus, it is postulated that RAGE mediates microglial activation by βA peptide by producing cytotoxic cytokines that cause neuronal damage in AD patients. In addition, RAGE was also demonstrated to specifically bind with βA peptide and mediate βA peptide-induced oxidative stress.
Cell receptors that bind to βA peptide have been identified. The low-affinity neurbtrophin receptor p75 (p75NTR) which belongs to the family of apoptotic receptors that generate cell-death signals on activation was found throughout the brains of AD patients. βA peptide was found to be a ligand for p75NTR, and to cause preferential apoplosis of neurons and normal neural crest-derived melanocytes that express p75NTR upon specifically binding to p75NTR.
Basal forebrain cholinergic neurons express the highest levels of p75NTR in the adult human brain and have been shown to be involved in AD. The expression of p75NTR by wild-type and mutant PC12 cells was shown to potentiate βA peptide-induced cell death. This interaction of βA peptide with p75NTR to mediate neuronal death in AD suggested a new target for therapeutic intervention.
Recently, ERAB which is overexpressed in neurons of the AD brain, was shown to bind with βA peptide to induce neuronal death in AD. Blocking ERAB with an antibody, anti-ERAB F(ab′)2, was found to reduce the βA peptide-induced cell death while ERAB overexpression increases βA peptide-induced cell death.
Nerve growth factor (NGF) is important for the survival and maintenance of central cholinergic neurons. Considerable evidence from animal studies suggests that NGF may be useful in reversing, halting, or at least slowing the progression of AD-related cholinergic basal forebrain atrophy. Administration of NGF was reported to attenuate degeneration of neurons and improve cognitive behavior in animals by stimulating central cholinergic neurons that are known to die during the development of AD. A clinical trial using intracranial infusion of NGF was reported to improve the patient's verbal episodic memory. Thus, attempting to counteract the degeneration of cholinergic neurons by NGF or compounds having neurotrophic properties may be a reasonable approach to treat AD. There are several different possible methods for stimulating NGF receptors, such as, NGF infusion, implantation of slow-release biodegradable pellets, using carrier-mediated transport across the blood-brain barrier, grafting NGF-producing cells, transferring genes directly to the brain, developing NGF receptor agonists, or controlling the endogenous NGF production.
In designing inhibitors of βA peptide toxicity, it was found that neither the alteration of the apparent secondary structure of βA peptide nor the prevention of βA peptide aggregation is required to abrogate the cytotoxicity of βA peptide. Nonetheless, inducing changes in aggregation kinetics and in higher order structural characteristics of βA peptide aggregates also proved to be effective in reducing βA peptide toxicity. See Sotoet al., Neuroreport 7, 721 (1996). Synthetic inhibitors that interact with βA peptide were shown to completely block βA peptide toxicity against PC12 cells, demonstrating that complete disruption of amyloid fibril formation is not necessary for abrogation of toxicity. It was also demonstrated that dipolar compounds such as phloretin and exifone that decrease the effective negative charge of membranes can prevent the association of βA peptide with negatively charged lipid vesicles and thereby prevent βA peptide-induced cytotoxicity. See Hertel et al., Proc. Nail. Acad. Sci. USA 94, 9412 (1997). These results suggest that βA peptide toxicity can be mediated through a physicochemical interaction with cell membranes.
There is strong interest in discovering potentially valuable natural products from natural sources for drug development. One reasonable source of such natural products involves medicinal plants that have been used throughout history for treating illness. Thus, the isolation of potentially valuable natural products from plants that can protect neurons from βA peptide insult is of interest. Although there are extensive reports that describe the extraction, fractionation, and chemical elucidation of natural products, plants have attracted relatively little attention as potentially valuable resources for drug discovery in the area of AD research. In order to achieve this goal, it is important to isolate and identify the chemical constituents in plants responsible for the biological activity.
Turmeric has been used as a curry spice and is used in traditional Indonesian medicine. Curcumin, a chemical constituent of turmeric, is an inhibitor of arachidonic acid metabolism and is a good anti-inflammatory agent. Curcumin is known to have antioxidative properties and has been shown to exhibit antitumor activity. Currently, curcumin is being evaluated as a chemopreventive agent by the National Cancer Institute.
G. biloba is a living fossil tree having undergone little evolutionary change over almost 200 million years. Extracts of the leaves have been used for 5,000 years in traditional Chinese medicine for various purposes. In 1994, a standardized dry extract of Ginkgo biloba leaves was approved by German health authorities for the treatment of primary degenerative dementia and vascular dementia. Currently, more than twenty four different brands of G. biloba extract are sold in the United States.
The use of complementary medications such as plant extracts in dementia therapy, varies according to different cultural traditions. In orthodox Western medicine, the pharmacological properties of traditional cognitive or memory enhancing plants have not been widely investigated in the context of current models of Alzheimer's disease. An exception is Ginkgo biloba L. in which the ginkgolides have been proposed to possess antioxidant, neuroprolective, and cholinergic activities relevant to Alzheimer's disease mechanisms. The leaves of G. biloba have been used as medicine for the treatment of peripheral or cerebral circulatory disorders, as well as for vascular and Alzheimer-type dementia. The therapeutic efficacy of G. biloba extracts in the treatment of Alzheimer's disease is reportedly similar to currently prescribed drugs such as tacrine or donepezil. See Maurer et al., J. Psychiatr. Res. 31, 645 (1997). In addition, the undesirable side effects of G. biloba are minimal. However, while there are more than two hundred articles published on the potential anti-AD effects of ginkgo products, the active constituents of the plant have not been isolated and identified.
Ginger is one of the world's favorite spices, and was probably discovered in the tropics of Southeast Asia. Ginger has benefitted humankind as a wonder drug since the beginning of recorded history.
It has been hypothesized that natural products capable of protecting neuronal cells from βA peptide insult can be discovered from plants, and specifically from turmeric, ginkgo biloba, and ginger. Although no anti-AD natural product derived drug that modulates βA peptide effect has been identified, historically, plants have been used for medicinal purposes that include alleviating the symptoms of AD. Among the medicinal plants suggested for the treatment of AD, ginkgo biloba and Huperzia serrata have been most extensively investigated. Huperzine A., a naturally occurring cholinesterase inhibitor from a moss Huperzia serrata is one natural product under development as a therapeutic agent to treat AD patients. See Skolnick, JAMA 277, 776 (1997). Further, a number of synthetic acetylcholinesterase inhibitors are under development as therapeutic agents against AD. However, while the published data indicate that the acetylcholinesterase inhibitor approach may be good for alleviating some of the symptoms of AD, such as improving memory, this approach does not cure or prevent the onset of the disease. Consequently, there remains a need to identify, isolate, and synthetically prepare new and improved anti-AD drugs which can provide chemotherapeutic and chemopreventive methods for the treatment of AD.